In the production of cell culture derived products, low maximum viable cell density and product expression levels result in high production costs. Birch, J. R., et al., "Large Scale Production of Monoclonal Antibodies" presented at Biotech '85 USA conference, Washington, D.C., Oct. 21-23, 1985. In traditional cell culture, cell growth and product expression are often limited due to the use of non-optimal culture media. Applicants have shown that product expression can be increased several fold by using media which contain synergistic combinations of nutrients (copending U.S. patent application "Cell Culture Medium for Enhanced Cell Growth, Culture Longevity and Product Expression", to Howarth, W., et al., U.S. Ser. No. 248,634 now abandoned). However, even in these superior media conditions, growth and product expression can still be optimized by further improving growth and product expression and thereby reducing production costs. The invention presented by the current patent application provides means for such optimization.
Ammonia has been shown to affect cells in culture in two ways. First, ammonia is a waste product of cellular metabolism which is toxic to the cells. Second, applicants have demonstrated a solute stress effect of ammonia whereby the per cell expression rate of secreted protein products can be increased in the presence of ammonia in the culture medium (but at the expense of a decrease in cell growth rate and/or maximum viable cell density) (U.S. patent application "Method of Increasing Product Expression through Solute Stress", to Maiorella, B., et al., U.S. Ser. No. 122,015, filed on Nov. 18, 1987 now abandoned).
In culture solution, ammonia can exist in the form of ammonia or ammonium ions. As used in this patent application, the word "ammonia" includes the above two forms. Ammonia is a normal waste product of amino acid metabolism and in particular of glutamine metabolism (Haussinger, D., Sies, H., Glutamine Metabolism in Mammalian Tissue, Springer Verlag, 1984; McKeehan, W. L. "Glycolysis, Glutaminolysis and Cell Proliferation", Cell Biology International Reports, 6(7):635- 650, 1982; Reuveny, S., et al., 1986, J. Immunol. Methods, 66: 53-59). It is also produced by the spontaneous degradation of glutamine in the medium (Ozturk, S. S., et al., 1990, Biotechnol. Progress, 6:121-128). When left to accumulate, it can reach a toxic level in the culture and cause the cessation of cell growth and eventually cell death (Holley, R. W., et al., 1978, PNAS (USA), 75(4):1864-1866). It can also adversely affect production of cell products (Reuveny, S., et al., 1986, "Factors Affecting Cell Growth and Monoclonal Antibody Production in Stirred Reactors", J. Immun. Methods, 86:53-59).
The solute stress effect of ammonia can be observed when ammonia is added in such concentration, at least above the concentration found optimal for cell growth but less than that which causes culture death. The per cell rate of product expression and/or the culture longevity of cells under solute stress is comparatively greater than in the absence of added ammonia.
Attempts at overcoming the toxic effect of ammonia have centered around the removal of ammonia from cell culture. For example, by means of continuous replenishment of the culture medium, computer controlled glutamine level (Glacken, M. W., et al., 1986, "Reduction of Waste Product Excretion via Nutrient Control", Biotech. Bioeng., 28:1376-1389), and continuous perfusion (Reuveny, S., et al., 1986, "Comparision of Cell Propagation Methods for their Effect on Monoclonal Antibody Yield in Fermentors", J. Immun. Methods, 86:61-69). Some researchers have also advocated minimizing the concentration of glutamine present in the culture by adapting the culture to grow in the absence of glutamine and with glutamic acid as an alternate substrate (Griffiths, J. B., et al., 1967, "The Uptake of Amino Acids by Mouse Cells During Growth in Batch Culture and Chemostat Culture, the Influence of Cell Growth Rate", Proc. Roy. Soc. B., 168:421-438). In other cases, glutamine is slowly added throughout the time course of the culture to maintain a relatively constant low concentration of glutamine (Glacken, M. W., et al., 1986, "Reduction of Waste Product Excretion via Nutrient Control: Possible Strategies for Maximizing Product and Cell Yields on Serum in Cultures of Mammalian Cells", Biotechnol. Bioeng., 28:1376-1389). However, these methods have proved unsatisfactory, due to for example, the complicated machinery entailed in the regulatory system.
Recently, investigations had been conducted regarding the toxicity of ammonia to mammalian cells in chemostat culture medium. These investigations employed the addition of ammonia and lactate to the medium in pulses and step changes (Miller, W. M., A Kinetic Analysis of Hybridoma Growth & Metabolism, U.C. Berkeley (Doctoral Dissertation, 1987); Miller, W. M., et al., 1988, Bioprocess Engineering, "Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture.") Miller et al., had also presented abstracts, "Transient and steady-state responses in continuous hybridoma culture--monoclonal antibody preparation", Abst. Pap. Am. Chem. Soc., MBTD 128, 1988; and "Kinetic analysis of transient responses in continuous hybridoma suspension--effect of culture medium composition" Abst. Pap. Am. Chem. Soc., MBTB 149, 1987.
The inhibitory ammonia concentration varies markedly between cell lines (Miller, W. M., et al., Bioprocess Engineering, supra). For example, the growth of mouse 3T3 cells was inhibited by less than 1 mM added ammonium chloride, whereas the growth of human HL-60 cells was inhibited at about 9 mM added ammonium chloride. Id. Miller et al., observed that a mouse hybridoma cell line AB2-143.2 gradually developed a tolerance to ammonia added in the feed to a continuous culture. It was originally observed that 5 mM of ammonia inhibited the growth of AB2-143.2 cell line. The cells were considered to have adapted to 8.2 mM of ammonia in the feed stream when in the culture fed with 8.2 mM ammonia they grew to the same cell concentration as non-adapted cells grown in medium without added ammonia (A Kinetic Analysis of Hybridoma Growth & Metabolism, supra. at p. 126; and Miller, W. M, et al., Bioprocess Engineering, supra). No data was presented on product expression. Miller et al., did not demonstrate nor show how cells can be adapted to acquire a phenotype by being exposed to ammonia such that the adapted cells would grow to a higher cell density and/or produce more cell products, as compared to their non-adapted counterparts, when both are subsequently grown in a culture medium without an initial added ammonia or with added ammonia at a level lower than that which the cells have been exposed to during the adaptation process.